research communications Acta Crystallographica Section F

Structural Biology Communications ISSN 2053-230X

Sompong Sansenya,a,b Risa Mutoh,c Ratana Charoenwattanasatien,a Genji Kurisuc and James R. Ketudat Cairnsa,d* a

Institute of Science, School of Biochemistry and Center for Biomolecular Structure, Function and Application, Suranaree University of Technology, 111 University Avenue, Nakhon Ratchasima Muang District, Nakhon Ratchasima 30000, Thailand, bDepartment of Chemistry, Faculty of Science, Rajamangala University of Technology, Thanyaburi, 39 Moo 1, Rangsit-Nakhon Nayok Road, Klong 6, Thanyaburi, Pathum Thani 12110, Thailand, c Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871, Japan, and dLaboratory of Biochemistry, Chulabhorn Research Institute, Vipavadee-Rangsit Highway, Bangkok 10210, Thailand

Correspondence e-mail: [email protected]

Received 3 November 2014 Accepted 20 November 2014

# 2015 International Union of Crystallography All rights reserved

Acta Cryst. (2015). F71, 41–44

Expression and crystallization of a bacterial glycoside hydrolase family 116 b-glucosidase from Thermoanaerobacterium xylanolyticum The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metalaffinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS–PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pNP) glycosides pNP- -dglucoside, pNP- -d-galactoside and pNP-N-acetyl- -d-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 M sodium citrate tribasic, 0.1 M Tris–HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions ˚ of 120  25  5 mm. The TxGH116 crystals diffracted X-rays to 3.15 A resolution and belonged to the monoclinic space group P21. Structure solution will allow a structural explanation of the effects of human GBA2 mutations.

1. Introduction Glycoside hydrolase family 116 (GH116) includes animal, plant, archaeal and bacterial protein sequences similar to human bile acid -glucosidase, which was found to be the nonlysosomal glucosylceramidase or -glucosidase 2 and designated GBA2 (Matern et al., 2001; Yildiz et al., 2006; Boot et al., 2007; Cobucci-Ponzano et al., 2010). GBA2 has recently become of interest owing to its involvement in genetic disorders leading to paraplegia (Hammer et al., 2013; Martin et al., 2013). Currently, the characterization of GH116 is limited, with recombinant expression and characterization having been reported for human GBA2 (Matern et al., 2001), the Sulfolobus solfataricus -xylosidase/ -glucosidase SSO1353 (Cobucci-Ponzano et al., 2010; Ko¨rschen et al., 2013) and the S. solfataricus -N-acetylglucosaminidase/ -glucosidase SSO3013 (Ferrara et al., 2014), while bacterial enzymes have not been described to date. The human enzyme is known to hydrolyze glycosides of the steroid-related bile acids (Matern et al., 1992, 1997, 2001) and glucosylceramide (Yildiz et al., 2006; Boot et al., 2007). The catalytic nucleophile of SSO1353 has been identified by labelling with the mechanism-based inhibitor 2,4dinitrophenyl -d-2-deoxy-2-fluoroglucoside and the acid/base by mutation and nucleophilic rescue experiments (Cobucci-Ponzano et al., 2010), but other structural features related to catalysis by GH116 enzymes await a crystal structure. Recently, knockout of GBA2 has been identified to be of benefit in a mouse model of Gaucher disease (which results from a deficiency of the lysosomal -glucosidase glucosylceramidase 1, also called GBA), so inhibitors of GBA2 may serve as possible treatments for Gaucher disease (Mistry et al., 2014). Although inhibitors of GBA2 have been characterized (Overkleeft et al., 1998; Ridley et al., 2013), the development of more specific inhibitors may benefit from greater structural knowledge. Therefore, a GH116 protein from the rod-shaped, spore-forming anaerobic thermophile Thermoanaerobactirium xylanolyticum has been identified as a thermostable bacterial enzyme that could be readily crystallized to produce a structural model for GH116. doi:10.1107/S2053230X14025461

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research communications Table 1

Table 2

Production of TxGH116.

Crystallization.

Source organism DNA source 50 sequence 30 sequence Cloning vector Expression vector Expression host Complete amino-acid sequence of the construct produced

T. xylanolyticum Synthesized at GenScript NcoI site: CCATGGCG Stop codon and XhoI site: TAAGCTTCTCGAG pUC57 pET-30a E. coli strain BL21(DE3)

Method Plate type Temperature (K) Protein concentration (mg ml1) Buffer composition of protein solution Composition of reservoir solution

MHHHHHHSSGLVPRGSGMKETAAAKFERQHMDSPDLGTDDDDKAMAMRKVIILLFIFIFTISAILTGCSEKININEDKISHKIDIPDSAWTIGIGEKFKNAGHPNVKYPMIDDSYVQGAPLGGFGAGTIGRTYNGGFSRWHLEIGKNKYTTVYANQFSVFQKVEGNKDGVAQVLYAGEPENGYLSSWKWDYPKESGMYYALYPNSWYTYTNKDLPVQLAVKQFSPIIPYNYKETSYPVAVFKWTAYNPTNKNVDVSIMFTWQNMIGFFGKQVNVNSGNFNKIIKDKSKDSEIVAAVMGNISNDNEEWNGEYSIGVKKVPGVDISYKAKFVTTGDGSDLWHEFSKNGILDNKDDETPTKQDGIGSAIAVNFKLQPGQTIEVPFALSWDLPIMKFGGGDKWYKMYTKYFGKNGKNSFAILKEALNNYQKWEKMIDDWQKPILSNKSKPDWYKTALFNELYYLADGGTAWENGKVGEKDKRTNNMFGLLECFDYNYYETLDVRFYGSFPLVMLWPDIEKQVMRQFADTINVQDSSEFKVGSNGAMAVKKVQGMIPHDLGSSYALPWIKINAYDWQNPNIWKDLNSKYVLLVYRDYVLTGKTDKEFLKYTWKSVKTALDKLKEMDKDNDGIPDNEGIPDQTYDTWSMKGTSAYCGSLWLAALKAAQEIGKVLKDNEAYIKYNEWYKIAQQNFEKELWNGEYYNFDTESDHKDSIMADQLAGQWYADILRLGDILPKDHVQKALKKIYEFNVMKFENGKMGAVNGMRPDGIVDESDIQAQEVWTGVTYALASFMKYRGMTEEAYNTAYGVYKMTYDKSGKGYWFRTPEAWTKDGNYRASMYMRPLSIWSMEVNYNEV

Volume and ratio of drop Volume of reservoir (ml)

2. Materials and methods 2.1. Recombinant TxGH116 production

A gene (GenBank accession No. KM677956) encoding the GH116 protein from T. xylanolyticum (GenBank accession No. AEF18218.1) optimized for expression in Escherichia coli and synthesized by GenScript was cloned into the NcoI and XhoI sites of pET-30a(+) and the sequence was confirmed by automated DNA sequencing. The recombinant plasmid (pET-30aTxGH116) was transformed into E. coli strain BL21(DE3), which was grown in LB medium with 50 mg ml1 kanamycin and incubated at 310 K (shaking at 200 rev min1) until the optical density at 600 nm reached 0.6. Isopropyl -d-1-thiogalactopyranoside (IPTG) was added to 0.3 mM and the culture was grown at 293 K for 18 h. The cells were pelleted by centrifugation and stored at 193 K. Cell pellets were suspended in 5 ml extraction buffer [20 mM Tris– HCl pH 7.5, 200 mg ml1 lysozyme, 1% Triton X-100, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM 6-aminohexanoic acid, 1 mM benzamidine hydrochloride] per gram, incubated at room temperature for 30 min and sonicated at 277 K for 15 min. After centrifugation at 164 000g for 15 min at 277 K, the protein solution was heated for 30 min at 323 and 348 K. The supernatant was applied onto an Ni–IMAC column pre-equilibrated with equilibration buffer (20 mM Tris–HCl pH 7.5, 50 mM NaCl) at 277 K. The column was washed and eluted with 5, 10 and 250 mM imidazole in equilibration buffer. All fractions were assayed for the hydrolysis of 5 mM p-nitrophenyl -dglucopyranoside (pNPGlc) in 50 mM phosphate buffer pH 6.0, and the protein composition was assessed with SDS–PAGE. Selected fractions were pooled and concentrated, and loaded onto a Superdex 200 gel-filtration column (GE Healthcare) equilibrated and eluted with 20 mM Tris–HCl pH 7.5, 150 mM NaCl. The purified protein was concentrated and stored at 277 K. Macromolecule-production information is summarized in Table 1. 2.2. Crystallization

The initial crystallization of TxGH116 was screened by hangingdrop vapour diffusion using the SaltRx and SaltRx 2 screens

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Vapour diffusion Hanging drop 293 10 20 mM Tris–HCl pH 7.5 0.6 M sodium citrate tribasic, 0.1 M Tris–HCl pH 7.0 2 ml protein:1 ml precipitant 150

Table 3 Data collection and processing. Values in parentheses are for the outer shell. Diffraction source ˚) Wavelength (A Temperature (K) Detector Crystal-to-detector distance (mm) Rotation range per image ( ) Total rotation range ( ) Exposure time per image (s) Space group ˚ , ) Unit-cell parameters (A Mosaicity ( ) ˚) Resolution range (A Total No. of reflections No. of unique reflections Completeness (%) Multiplicity hI/(I)i Rr.i.m.† (%) ˚ 2) Overall B factor from Wilson plot (A

BL44XL, SPring-8 0.90 100 PX210 detector (Rayonix, USA) 300 0.5 180 5 P21 a = 56.2, b = 169.0, c = 234.8,  = = 90, = 94.6 0.487–1.183 50–3.15 (3.20–3.15) 278686 75125 99.9 (100) 3.7 (3.7) 9.35 (2.67) 15.5 (46.6) 34.797

† The redundancy-independent merging R factor Rr.i.m. was estimated by multiplying the linear Rmerge value by the factor [N/(N  1)]1/2, where N is the data multiplicity.

(Hampton Research). The purified TxGH116 (approximately 90% purity) was adjusted to 7–10 mg ml1 in 20 mM Tris–HCl pH 7.5 by centrifugal filtration. The hanging-drop vapour-diffusion plate contained 150 ml precipitant in the reservoir, and the drops consisted of 2 ml protein solution and 1 ml precipitant solution. All crystallization experiments were carried out at 293 K. Clusters of small crystals were observed in several conditions within 10 d. The conditions consisting of 0.7 and 1.2 M sodium citrate tribasic, 0.1 M Tris– HCl pH 8.0, which gave small single crystals, were used for optimization. Optimization was accomplished by varying the pH from 7.0 to 8.5 and the salt concentration from 0.2 to 2 M and using protein concentrations of 5 and 10 mg ml1 to arrive at the conditions shown in Table 2. Needle clusters and large crystals were harvested from the original crystallization drop and washed with precipitant solution six times. A preparation of these washed crystals was crushed with a nylon loop in 100 ml precipitant and serially diluted 10, 100 and 1000 times. The diluted crystal seed stocks were streaked into crystallization drops that had been pre-incubated for 30 min at 293 K to produce large single crystals. Another preparation of crystals was dissolved in 1 SDS–PAGE buffer in 0.8 precipitant and analyzed by SDS–PAGE to verify the protein form in the crystals.

2.3. Data collection and processing

X-ray diffraction data were collected from TxGH116 crystals on the BL44XL beamline at the SPring-8 synchrotron, Japan. Before diffraction, the crystals were soaked in a cryoprotectant solution (3.0 M sodium malonate in 0.6 M tribasic sodium citrate, 0.1 M Tris– HCl pH 7.0) for 15 min and then flash-vitrified in liquid nitrogen. As shown in Table 3, data sets were collected at 100 K using a Acta Cryst. (2015). F71, 41–44

research communications CCD-based PX210 detector system and at 0.5 oscillations (5 s each), with a crystal-to-detector distance of 300 mm. The data were processed with the HKL-2000 suite (Otwinowski & Minor, 1997).

3. Results and discussion

Figure 1 SDS–PAGE analysis of purification fractions and protein crystals. (a) SDS–PAGE of the extraction, heating and IMAC steps. Lane 1, soluble fraction from crude cell extract; lane 2, extract after heating at 323 K; lane 3, extract after heating at 348 K; lane 4, flowthrough of the IMAC column; lane 5, wash of the IMAC column with equilibration buffer; lane 6, wash with 5 mM imidazole; lanes 7 and 8, wash with 10 mM imidazole; lane 9, elution with 250 mM imidazole. (b) SDS–PAGE of the ten Superdex 200 fractions that were pooled for crystallization. (c) SDS–PAGE of dissolved clustered crystals from the same condition as the diffracted crystal. The crystal clusters were washed six times with precipitant before dissolving them in 1 SDS–PAGE sample buffer in 0.8 precipitant solution. Lane P contains the protein crystal. In all cases, lane M contains molecular-weight marker with masses in kDa shown to the left. The gel was 11% polyacrylamide run in Tris–glycine buffer and stained with Coomassie Brilliant Blue R250 using standard methods.

The T. xylanolyticum protein entry AEF18218.1, designated as TxGH116, was identified as a possible thermophilic GH116 structural model homologous to the human GBA2 protein sequence (NP_065995), with which it shares 37% amino-acid sequence identity over 783 residues. The full-length TxGH116 sequence contains 806 amino-acid residues, and is more similar to the eukaryotic proteins from animals and plants than to the archaeal GH116 enzymes (Supplementary Fig. S1). TxGH116 was expressed as an N-terminally His6-tagged fusion protein of 852 amino acids (Table 1), with a calculated mass of 97.5 kDa, in E. coli strain BL21(DE3) cells. The protein bound poorly to the IMAC column, so the fractions that eluted in 5–10 mM imidazole with >70% pure protein were used for size-exclusion chromatography, from which >90% pure protein was obtained (Fig. 1). Since the major protein band ran below the 90 kDa marker on SDS–PAGE, which is significantly lower than the expected 97 kDa for the full-length fusion protein, the poor IMAC binding suggests that the N-terminal tag may have been lost to proteolysis. TxGH116 is a thermophilic -d-glucosidase, with weaker -galactosidase and N-acetyl- -glucosaminidase activities, and has a pH optimum of 6.0 and a temperature optimum of 358 K (Supplementary Table S1, Figs. S2 and S3), which are appropriate for the growth conditions of T. xylanolyticum (Lee et al., 1993). Optimized crystals with maximum dimensions of 120  25  5 mm were obtained using a precipitant consisting of 0.6 M tribasic sodium citrate, 0.1 M Tris–HCl pH 7.0 (Table 2) with microseeding within 2–5 d (Fig. 2). The crystals contained the 90 kDa protein, as shown by SDS–PAGE (Fig. 1c). Table 3 shows the statistics for the best diffraction data that were obtained from crystals of TxGH116, which ˚ (Supplementary Fig. S4). had a nominal resolution of 3.15 A The addition of a bacterial -glucosidase to the previously characterized animal GBA2 (Matern et al., 2001) and archaeal -xylosidase/ -glucosidase (Cobucci-Ponzano et al., 2010) and N-acetyl -glucosaminidase (Ferrara et al., 2014) expands the description of GH116 function, and the forthcoming crystal structure will serve as a model for GH116 enzymes, including human GBA2. We are continuing our efforts to improve crystal diffraction and to produce selenomethionine-labelled protein crystals to allow solution of the structure by single-wavelength or multiple-wavelength anomalous dispersion, since no GH116 structural models have yet been published. This work was supported by Suranaree University of Technology via the National Research University Project of the Commission on Higher Education of Thailand, the Institute for Protein Research (IPR) of Osaka University and the Thailand Research Fund Grant BRG5680012. Data collection at the SPring-8 beamline was supported by the Cooperative Research Program of the IPR, Osaka University.

References Figure 2 Crystals from the condition used for diffraction. The crystals were obtained in a drop consisting of 1 ml 0.6 M tribasic sodium citrate, 0.1 M Tris–HCl pH 7.0 mixed with 2 ml 10 mg ml1 protein solution with microseeding.

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